Abstract

1514

Introduction: Syk is a non-receptor tyrosine kinase widely expressed in the hematopoietic system, where it regulates the related activities of proliferation and survival as well as actin cytoskeleton-mediated adhesion and motility changes. Recently, Syk was shown to down-regulate Epidermal Growth Factor Receptor (EGFR)-mediated responses such as proliferation and survival in a non-tumorigenic cell line, but was not able to counteract the activity of another member of the EGFR family, HER2/neu (ErbB2). HER2/neu is found overexpressed in about 30% of human breast and ovarian cancers and is associated with poor clinical outcome. After dimerization, HER2/neu activates signal transduction pathways that favor tumorigenesis via dysregulation of proliferation, survival, cell migration and adhesion. We are currently screening breast cancer cell lines by western blot for their expression of EGFR, HER2/neu and Syk in order to determine whether a potential link between Syk and HER2/neu exists in EGFR-negative cancer cell lines. Methods: A total of 9 cell lines were chosen for this study: MCF7, DU4475, BT474, MDA-MB 330, MDA-MB 468, MDA-MB 361, MDA-MB 453, AU565 and HMEC. Cells were trypsinized, spun down, washed in PBS and frozen in liquid nitrogen until used. Frozen pellets were resuspended in lysis buffer, incubated on ice for 15 minutes and centrifuged. Western blots were performed with antibodies against EGFR, Syk and HER2/neu on supernatants from total cell lysates. Band intensity was measured by image analysis software with actin as loading control. Results: Thus far, 8 human breast epithelial cancer cell lines have been screened for expression of EGFR, Syk and HER2/neu protein. Normal human mammary epithelial cells, HMEC, were used as positive control for Syk expression and negative control for HER2/neu over-expression. With the exception of MDA-MB-468, none of the cell lines tested expressed EGFR. Of the remaining 7 breast cancer cell lines, MCF7 and DU4475 showed high expression of Syk and low or no expression of HER2/neu. The opposite was true for MDA-MB453, AU565, MDA-MB-330 and MDA-MB-361. Only one cell line, BT474, was positive for both Syk and HER2/neu. Conclusion: We tested 9 breast epithelial cell lines for the expression of EGFR, Syk tyrosine kinase and HER2/neu. Of the nine cell lines tested, only one expressed EGFR. An inverse relationship was found in 7 EGFR negative cancer cases between Syk and HER2/neu whereby if Syk was highly expressed HER2/neu was absent, while if HER2/neu was overexpressed Syk was not present or present at very low levels. Only one cell line (EGFR negative) showed concomitant expression of both Syk and HER2/neu. Our in vitro observation of a reverse correlation between HER2/neu and Syk in EGFR negative cell lines may suggest an interesting link between these two molecules.